The brain structures and neural circuits regulating anxiety behavior are poorly understood. The hippocampus is thought to contribute to anxiety via its role in resolving conflicting goal-directed behavior as part of the behavioral inhibition system. Projections from the hippocampus to the medial prefrontal cortex carry information about the anxiety state of the animal and are proposed to be a critical conduit for the anxiety-modulating function of the hippocampus. However, until now it has not been possible to dissect the contributions of the hippocampal projections to the frontal cortex because classical lesions and pharmacological manipulations of these structures interfere with the basal function of these structures. Here we propose the development and validation of a novel pharmacogenetic inhibition technology to selectively manipulate functional connections between brain structures in awake behaving animals. This novel tool is based on the selective targeting of an inhibitory GPCR to axon pre-terminals. Expression of this axonal inhibition tool in hippocampal projection neurons followed by tissue-specific delivery of the GPCR agonist into target tissues will allow disruption of selected hippocampal outputs without altering neural firing activity. We propose to develop and validate a human muscarinic M4 DREADD receptor/serotonin 1B receptor chimera in two specific aims. Aim 1 involves the construction of the receptor, and characterization of its cellular localization, as well as the construction of a viral vector capable of delivering the chimeric receptor to neurons in vivo. Aim 2 involves the use of this vector to test the effects on evoked and spontaneous neural transmission in the ventral hippocampus-medial prefrontal cortex network. Successful completion of these experiments will prepare the applicants to apply this tool to dissect the role of selected hippocampal ouputs in regulating anxiety and other hippocampal-dependent behaviors, and will become proof-of-concept data to support a larger, R01 funding application. In the long run this work promises to establish a new pharmacogenetic neural connectivity inhibition tool that is likely to find wide use in the neuroscience field. PUBLIC HEALTH RELEVANCE: Developing improved treatments for anxiety requires greater knowledge of the neurobiology underlying this devastating set of illnesses. The current project seeks to develop a tool that will permit us to test the role of specific neural pathways in anxiety-related behavior. In the future, such a tool would be invaluable in identifying which neural pathways are required for anxiety, facilitating the development of new treatments.